scholarly journals Identification of Residues Responsible for the Defective Virulence Gene Regulator Mga Produced by a Natural Mutant of Streptococcus pyogenes

2005 ◽  
Vol 187 (17) ◽  
pp. 5955-5966 ◽  
Author(s):  
Cheryl M. Vahling ◽  
Kevin S. McIver
Keyword(s):  
Streptococcus Pyogenes ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Binding Activity ◽  
Human Pathogen ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Naturally Occurring ◽  
Electrophoretic Mobility Shift Assays ◽  
Regulated Promoters

ABSTRACT Mga is a transcriptional regulator in the pathogen Streptococcus pyogenes that positively activates several important virulence genes involved in colonization and immune evasion in the human host. A naturally occurring mutant of Mga that is defective in its ability to activate transcription has been identified in the serotype M50 strain B514-Sm. Sequence alignment of the defective M50 Mga with the fully functional Mga from serotypes M4 and M49 revealed only three amino acid changes that might result in a defective protein. Electrophoretic mobility shift assays using purified M50 and M4 maltose binding protein-Mga found that both exhibited DNA-binding activity towards regulated promoters. Thus, the significance of each residue for the functionality of M50 Mga was explored through introduction of “gain-of-function” mutations based on M4 Mga. Transcriptional studies of the mutant alleles under both constitutive (PrpsL) and autoactivated (Pmga4) promoters illustrated that an arginine-to-methionine change at position 461 of M50 Mga protein fully restored activation of downstream genes. Western blot analyses of steady-state Mga levels suggest that the M461 residue may play a role in overall conformation and protein stability of Mga. However, despite the conservation of the M461 protein among all other Mga proteins, it does not appear to be necessary for activity in a divergent M6 Mga. These studies highlight the potential differences that exist between divergent Mga proteins in this important human pathogen.

scholarly journals RAG-2 Promotes Heptamer Occupancy by RAG-1 in the Assembly of a V(D)J Initiation Complex

1999 ◽  
Vol 19 (5) ◽  
pp. 3674-3683 ◽  
Author(s):  
Patrick C. Swanson ◽  
Stephen Desiderio
Keyword(s):  
Electrophoretic Mobility ◽  
Binding Activity ◽  
Cross Linking ◽  
Mobility Shift ◽  
Initiation Complex ◽  
Dna Binding Activity ◽  
Recombination Signal Sequences ◽  
Rag 1 ◽  
Electrophoretic Mobility Shift Assays ◽  
Single Subunit

ABSTRACT V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.

scholarly journals The Bacteroides fragilis BtgA Mobilization Protein Binds to the oriT Region of pBFTM10

1998 ◽  
Vol 180 (18) ◽  
pp. 4922-4928 ◽  
Author(s):  
Leonid A. Sitailo ◽  
Alexander M. Zagariya ◽  
Patrick J. Arnold ◽  
Gayatri Vedantam ◽  
David W. Hecht
Keyword(s):  
Specific Binding ◽  
Bacteroides Fragilis ◽  
Binding Activity ◽  
Inverted Repeats ◽  
Mobility Shift ◽  
E Coli ◽  
Dna Binding Activity ◽  
Footprint Analysis ◽  
Nick Site ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Bacteroides fragilis conjugal plasmid pBFTM10 contains two genes, btgA and btgB, and a putative oriT region necessary for transfer inBacteroides fragilis and Escherichia coli. The BtgA protein was predicted to contain a helix-turn-helix motif, indicating possible DNA binding activity. DNA sequence analysis of the region immediately upstream of btgA revealed three sets of inverted repeats, potentially locating the oriTregion. A 304-bp DNA fragment comprising this putative oriT region was cloned and confirmed to be the functional pBFTM10 oriT by bacterial conjugation experiments using E. coli and B. fragilis. btgAwas cloned and overexpressed in E. coli, and the purified protein was used in electrophoretic mobility shift assays, demonstrating specific binding of BtgA protein to its cognateoriT. DNase I footprint analysis demonstrated that BtgA binds apparently in a single-stranded fashion to theoriT-containing fragment, overlapping inverted repeats I, II, and III and the putative nick site.

scholarly journals Autoregulation of hpdR and its effect on CDA biosynthesis in Streptomyces coelicolor

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2641-2648 ◽  
Author(s):  
Haihua Yang ◽  
Yang An ◽  
Linqi Wang ◽  
Shuli Zhang ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Promoter Region ◽  
Streptomyces Coelicolor ◽  
Binding Activity ◽  
Catabolic Gene ◽  
Mobility Shift ◽  
S1 Nuclease ◽  
Dna Binding Activity ◽  
Electrophoretic Mobility Shift Assays ◽  
Positive Effect

HpdR, an IclR-family regulator in Streptomyces coelicolor, is a substrate-dependent repressor for the tyrosine catabolic gene hppD. In this study, S1 nuclease protection assays revealed that hpdR is subject to a negative autoregulation. Purified HpdR showed specific DNA-binding activity for the promoter region of hpdR, indicating that the autoregulation of hpdR is performed directly. The disruption of hpdR led to reduced production of CDA by S. coelicolor J1501, suggesting a positive effect of hpdR on CDA biosynthesis. Electrophoretic mobility shift assays showed that HpdR specifically bound to the promoter region of hmaS (SCO3229 in the CDA gene cluster), encoding 4-hydroxymandelic acid synthase. Disruption of hmaS in J1501 abolished CDA production. It is possible that hpdR regulates CDA biosynthesis by controlling the transcription of hmaS.

scholarly journals The Myb domain of LUX ARRHYTHMO in complex with DNA: expression, purification and crystallization

2016 ◽  
Vol 72 (5) ◽  
pp. 356-361 ◽  
Author(s):  
Catarina S. Silva ◽  
Xuelei Lai ◽  
Max Nanao ◽  
Chloe Zubieta
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Mobility Shift ◽  
Data Set ◽  
Dna Binding Activity ◽  
Protein Dna Interactions ◽  
Reported Data ◽  
Electrophoretic Mobility Shift Assays ◽  
Myb Domain

LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock.Luxmutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein–DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using theMeshAndCollectworkflow available at the ESRF.

A mammalian factor that binds telomeric TTAGGG repeats in vitro

1992 ◽  
Vol 12 (11) ◽  
pp. 4834-4843 ◽  
Author(s):  
Z Zhong ◽  
L Shiue ◽  
S Kaplan ◽  
T de Lange
Keyword(s):  
Sodium Dodecyl ◽  
Binding Activity ◽  
Repeat Sequence ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Circular Dnas ◽  
Electrophoretic Mobility Shift Assays ◽  
Ttaggg Repeat

We have identified a DNA-binding activity with specificity for the TTAGGG repeat arrays found at mammalian telomeres. This factor, called TTAGGG repeat factor (TRF), is present in nuclear extracts of human, mouse, and monkey cells. TRF from HeLa cells was characterized in detail by electrophoretic mobility shift assays. It binds double-stranded TTAGGG repeats in linear and circular DNAs. Single-stranded repeats are not recognized. The optimal site for TRF appears to contain more than six contiguous TTAGGG repeats. Tandem arrays of TAGGG, TTTAGGG, TTTTAGGG, TTGGGG, and TTAGGC repeats do not bind TRF well, indicating that TRF preferentially recognizes the telomeric repeat sequence present at mammalian chromosome ends. The apparent molecular mass of this factor, based on recovery of TRF from sodium dodecyl sulfate-polyacrylamide gels, is approximately 50 kDa. We suggest that TRF binds along the length of mammalian telomeres.

scholarly journals A mammalian factor that binds telomeric TTAGGG repeats in vitro.

1992 ◽  
Vol 12 (11) ◽  
pp. 4834-4843 ◽  
Author(s):  
Z Zhong ◽  
L Shiue ◽  
S Kaplan ◽  
T de Lange
Keyword(s):  
Sodium Dodecyl ◽  
Binding Activity ◽  
Repeat Sequence ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Circular Dnas ◽  
Electrophoretic Mobility Shift Assays ◽  
Ttaggg Repeat

We have identified a DNA-binding activity with specificity for the TTAGGG repeat arrays found at mammalian telomeres. This factor, called TTAGGG repeat factor (TRF), is present in nuclear extracts of human, mouse, and monkey cells. TRF from HeLa cells was characterized in detail by electrophoretic mobility shift assays. It binds double-stranded TTAGGG repeats in linear and circular DNAs. Single-stranded repeats are not recognized. The optimal site for TRF appears to contain more than six contiguous TTAGGG repeats. Tandem arrays of TAGGG, TTTAGGG, TTTTAGGG, TTGGGG, and TTAGGC repeats do not bind TRF well, indicating that TRF preferentially recognizes the telomeric repeat sequence present at mammalian chromosome ends. The apparent molecular mass of this factor, based on recovery of TRF from sodium dodecyl sulfate-polyacrylamide gels, is approximately 50 kDa. We suggest that TRF binds along the length of mammalian telomeres.

An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

Blood ◽  
2001 ◽  
Vol 98 (8) ◽  
pp. 2555-2562 ◽  
Author(s):  
Mark Loyevsky ◽  
Timothy LaVaute ◽  
Charles R. Allerson ◽  
Robert Stearman ◽  
Olakunle O. Kassim ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Regulatory Protein ◽  
Protein S ◽  
Fold Increase ◽  
Binding Activity ◽  
Mobility Shift ◽  
Iron Responsive Element ◽  
Element Binding Protein ◽  
Responsive Element ◽  
Electrophoretic Mobility Shift Assays

Abstract This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

scholarly journals Drosophila Mi-2 Negatively Regulates dDREF by Inhibiting Its DNA-Binding Activity

2002 ◽  
Vol 22 (14) ◽  
pp. 5182-5193 ◽  
Author(s):  
Fumiko Hirose ◽  
Nobuko Ohshima ◽  
Eun-Jeong Kwon ◽  
Hideki Yoshida ◽  
Masamitsu Yamaguchi
Keyword(s):  
Dna Binding ◽  
Polytene Chromosomes ◽  
Ectopic Expression ◽  
Binding Activity ◽  
Mobility Shift ◽  
Interacting Protein ◽  
Reciprocal Regulation ◽  
Dna Binding Activity ◽  
Expression Of Genes ◽  
Biochemical Analyses

ABSTRACT Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5′-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.

Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells

1992 ◽  
Vol 12 (9) ◽  
pp. 4104-4111
Author(s):  
L Sistonen ◽  
K D Sarge ◽  
B Phillips ◽  
K Abravaya ◽  
R I Morimoto
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Hsp70 Gene ◽  
Mobility Shift ◽  
Heat Shock Element ◽  
Dna Binding Activity ◽  
Erythroleukemia Cells

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

scholarly journals Free Fatty Acids Interfere with the DNA Binding Activity of the Virulence Regulator PrfA of Listeria monocytogenes

2020 ◽  
Vol 202 (15) ◽  
Author(s):  
Patrícia T. dos Santos ◽  
Rikke S. S. Thomasen ◽  
Mathias S. Green ◽  
Nils J. Færgeman ◽  
Birgitte H. Kallipolitis
Keyword(s):  
Fatty Acids ◽  
Dna Binding ◽  
Free Fatty Acids ◽  
Antimicrobial Agents ◽  
Virulence Gene ◽  
Binding Activity ◽  
Content Type ◽  
Dna Binding Activity ◽  
Virulence Regulator ◽  
Long Chain

ABSTRACT Naturally occurring free fatty acids (FFAs) are recognized as potent antimicrobial agents that also affect the production of virulence factors in bacterial pathogens. In the foodborne pathogen Listeria monocytogenes, some medium- and long-chain FFAs act as antimicrobial agents as well as signaling compounds, causing a repression of transcription of virulence genes. We previously observed that the master virulence regulator PrfA is involved in both the antimicrobial and virulence-inhibitory response of L. monocytogenes to selected FFAs, but the underlying mechanisms are presently unknown. Here, we present a systematic analysis of the antimicrobial and PrfA-inhibitory activities of medium- and long-chain FFAs of various carbon chain lengths and degrees of saturation. We observed that exposure to specific antimicrobial and nonantimicrobial FFAs prevented PrfA-dependent activation of virulence gene transcription and reduced the levels of PrfA-regulated virulence factors. Thus, an antimicrobial activity was not compulsory for the PrfA-inhibitory ability of an FFA. In vitro binding experiments revealed that PrfA-inhibitory FFAs were also able to prevent the constitutively active variant PrfA* from binding to the PrfA box in the promoter region of the virulence gene hly, whereas noninhibitory FFAs did not affect its ability to bind DNA. Notably, the unsaturated FFAs inhibited the DNA binding activity of PrfA* most efficiently. Altogether, our findings support a model in which specific FFAs orchestrate a generalized reduction of the virulence potential of L. monocytogenes by directly targeting the key virulence regulator PrfA. IMPORTANCE Listeria monocytogenes is a Gram-positive pathogen able to cause foodborne infections in humans and animals. Key virulence genes in L. monocytogenes are activated by the transcription regulator PrfA, a DNA binding protein belonging to the CRP/FNR family. Various signals from the environment are known to affect the activity of PrfA, either positively or negatively. Recently, we found that specific medium- and long-chain free fatty acids act as antimicrobial agents as well as signaling compounds in L. monocytogenes. Here, we show that both antimicrobial and nonantimicrobial free fatty acids inhibit PrfA-dependent activation of virulence gene transcription by interfering with the DNA binding activity of PrfA. Our findings suggest that free fatty acids could be candidates for alternative therapies against L. monocytogenes.

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